Two RpoT genes of Physcomitrella patens encode phage-type RNA polymerases with dual targeting to mitochondria and plastids

Angiosperms possess a small family of phage-type RNA polymerase genes that arose by gene duplication from an ancestral gene encoding the mitochondrial RNA polymerase. We have isolated and sequenced the genes and cDNAs encoding two phage-type RNA polymerases, PpRpoT1 and PpRpoT2, from the moss Physcomitrella patens. PpRpoT1 comprises 19 exons and 18 introns, PpRpoT2 contains two additional introns. The N-terminal transit peptides of both polymerases are shown to confer dual-targeting of green fluorescent protein fusions to mitochondria and plastids. In vitro translation of the cDNAs revealed initiation of translation at two in-frame AUG start codons. Translation from the first methionine gives rise to a plastid-targeted polymerase, whereas initiation from the second methionine results in exclusively mitochondrial-targeted protein. Thus, dual-targeting of Physcomitrella RpoT is caused by and might be regulated by multiple translational starts. In phylogenetic analyses, the Physcomitrella RpoT polymerases form a sister group to all other phage-type polymerases of land plants. The two genes result from a gene duplication event that occurred independently from the one which led to the organellar polymerases with mitochondrial or plastid targeting properties in angiosperms. Yet, according to their conserved exon-intron structures they are representatives of the molecular evolutionary line leading to the RpoT genes of higher land plants.     

Expression patterns of cotton chloroplast genes during development: implications for development of plastid transformation vectors

Although most plastid transformation studies have focused on chloroplast expression, plastid transformation can also be used to express genes in plastids of a wide variety of plant tissues by using appropriate plastid promoters. Based on the sequence of the Gossypium hirsutum chloroplast genome, we developed primers and amplified segments of 20 different plastid genes. The PCR products were labeled and used in filter dot blot hybridization studies to characterize their expression levels and patterns in total RNA isolated from light- and dark-grown cotton tissues at different developmental stages. A subset of 6 genes among these was further characterized by real time PCR. Highest expression levels were observed for rrn16 and psbA. Four genes were expressed in all samples at relatively constant levels: accD, atpA, matK and rrn16. Expression in root tissue was generally low. The results of our study can be used to predict which operons and promoters are most likely to be preferentially expressed in the plastids of tissues of interest at levels that would result in the desired phenotype, facilitating the development of plastid transformation vectors.